Tissue scaffolds for use in muscoloskeletal repairs

ABSTRACT

The present invention includes a tissue scaffold suitable for use in repair and/or regeneration of musculoskeletal tissue when implanted in a body that includes a porous substrate made from a biodegradable, hydrophobic polymer having a modified hyaluronic acid complex dispersed on the surface of and/or, where the modified hyaluronic acid complex is insoluble in water, yet soluble in mixtures of organic and aqueous solvents in which the hydrophobic polymer is soluble.

FIELD OF THE INVENTION

The present invention is directed to tissue scaffolds suitable for usein tissue repair and/or regeneration and that are composed of a poroussubstrate made from a biodegradable, synthetic polymer and that have ahydrophobic hyaluronic acid complex incorporated therewith.

BACKGROUND OF THE INVENTION

There is a clinical need for biocompatible and biodegradable structuralmatrices that facilitate tissue infiltration to repair/regeneratediseased or damaged tissue. Previous attempts have used a number ofnaturally occurring, as well as synthetic biodegradable materials asscaffolds in the tissue repair process.

One class of biodegradable and biocompatible materials is the family ofpolysaccharides. One particular polysaccharide used for scaffolds ishyaluronic acid (HA). HA is a naturally-occurring linear polysaccharidecomprised of D-glucuronic acid and N-acetyl-D-glucosamine. Thepolyanionic polymer has a range of molecular weight of from1,000-10,000,000 Daltons. The use of purified HA has become commonpractice for treatments such as corneal transplantation,viscosupplementation of the knee, anti-adhesive barriers in spinalsurgeries and wound healing applications.

HA has been explored as a scaffold to enhance the repair of tissue.However, the poor physical properties of natural HA and its rapidresorption by the body has restricted its use to applications where astructurally sound scaffold is not required.

Numerous groups have attempted to modify HA to obtain a more robust andwater insoluble biopolymer for tissue engineering applications. One lineof modifications involved the use of cross-linking agents. Thesecross-linking agents include formaldehyde, glutaraldehyde, vinylsulphone, biscarbodiimides, poly-functional epoxides, glycidyl ether,photocurable cinnamic acid derivatives and adipic dihydrazide. However,they are concerns regarding the potential toxicity of some of thecross-linking agents.

Alternatively, HA derivatives have been obtained by targeting specificfunctional groups of HA, such as carboxyl, hydroxyl and N-acetyl groupsthrough chemical reactions, such as esterification, acylation,amidation, and sulphation. An example of an esterified-HA is a productsold under the tradename HYAFF 11 (Fidia Advanced Biopolymers, AbanoTerme, Italy), which has all of its carboxyl groups esterified withbenzyl esters. This material is stable in aqueous media for a few weeks,but completely degrades within 2 months.

In many tissue repair applications, e.g. musculoskeletal tissue repairand/or regeneration, the scaffold must be structurally intact forperiods far longer than two months. While the use of HA in tissue repairis desired due to HA's function in extracellular matrices and itsrecognition by certain cell receptors that regulate attachment,proliferation, differentiation and matrix synthesis of certain celltypes, thus far approaches using natural HA or known modified-HA haveproven inadequate to provide tissue scaffolds that maintain thestructural integrity for extended periods of time that is necessary inapplications such as musculoskeletal tissue repair or regeneration. Theinventions described herein provide a solution to the need for suchtissue scaffolds.

SUMMARY OF THE INVENTION

The present invention is directed to a tissue scaffold suitable for usein repair and/or regeneration of musculoskeletal tissue. The scaffoldcomprises a biodegradable, porous substrate comprising a biodegradable,hydrophobic polymer and a hyaluronic acid complex incorporated with thesubstrate, wherein the hyaluronic acid complex is insoluble in water,yet soluble in mixtures of organic and aqueous solvents in which theselected hydrophobic polymer is soluble. The structures of thesubstrates in the present invention may be nonwoven, fibrous mats orporous foams. The nonwoven, fibrous mats comprise fiber composed ofbiodegradable, hydrophobic polymers. The foams also are composed ofbiodegradable, hydrophobic polymers. The hyaluronic acid complex may bedispersed and incorporated on surface(s) of the substrate and/ordispersed and incorporated throughout the substrate.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a scanning electron micrograph(SEM) cross-sectional image of atissue scaffold of the present invention.

FIG. 2 is a SEM cross-sectional image of a tissue scaffold of thepresent invention.

FIG. 3 shows Safranin-O (SO) histological sections (100×) of a tissuescaffold of the present invention including cultured bovinechondrocytes.

DETAILED DESCRIPTION OF THE INVENTION

In treatments such as musculoskeletal tissue repair and/or regeneration,tissue scaffolds must maintain their physical and structural integrityonce implanted into the body for extended periods of time, e.g. greaterthan two months, as compared to other treatments, e.g. cornealtransplantation, viscosupplementation of the knee, anti-adhesivebarriers in spinal surgeries and wound healing applications, where suchlong-term properties are not required. In addition, in order to providesuch long-term tissue scaffolds with long-term enhanced cell-growthproperties, hyaluronic acid (HA) material also must be able to remainincorporated with the scaffold for similar extended periods of time.Hydrophilic HA materials will dissipate relatively quickly when exposedto fluids of the body when implanted therein. As such, hydrophilic HAmaterials are not successful candidates for use in tissue scaffoldsintended for use in applications requiring physical integrity overextended periods of time.

In order to circumvent the shortcomings of tissue scaffolds usingunmodified HA or hydrophilic modified HA, this invention utilizes astructurally robust scaffold based on a porous substrate made frombiodegradable, hydrophobic polymers and a relatively hydrophobic HAcomplex, as further described herein, distributed and incorporated onand/or throughout the scaffold. The hydrophobic HA complexes used in thepresent invention are more compatible with the hydrophobic polymers thanunmodified HA.

As will be noted herein, the HA complex should be distributed on thesurfaces of and/or throughout the porous substrate to obtain optimumcell-growth properties in the tissue scaffold. In addition, the HAcomplex is incorporated with the porous substrate, such that HA complexis bound to the porous substrate and not readily removable from thesubstrate once implanted in the body.

In one embodiment, tissue scaffolds of the present invention utilize anHA complex that can be dissolved in an aqueous/organic solvent mixtureand blended with a solution of the hydrophobic polymer in an appropriateorganic solvent. The blend of the dissolved HA complex and hydrophobicpolymer subsequently can be lyophilized to generate a porous foam tissuescaffold having the HA complex dispersed and incorporated throughout thefoam scaffold substrate. Alternatively, the HA complex of the presentinvention can be dissolved in an organic/aqueous mixture thatsubsequently can be poured over a hydrophobic substrate, e.g. a fibrous,nonwoven mat, allowed to penetrate the substrate, and then lyophilizedto yield a substrate having the HA complex tightly adhered thereto.

As noted above, native HA is a hydrophilic, highly water-solublematerial. Therefore, in order to be used in the present invention,native HA must be modified in order to make it more compatible withhydrophobic, polymers utilized in the present invention. The HA complexof the present invention is insoluble in water and shows improvedsolubility in selected organic solvents and organic/aqueous solventblends as compared to native HA or hydrophilic modified HA. It is thischaracteristic of the modified HA that allows it to be more compatiblewith hydrophobic polymers as compared to highly hydrophilic native HA.

To achieve this compatibility, native HA was modified by two methods. Inthe first method, a macromolecular salt complex of HA was formed via theion-exchange of alkali metal monovalent salt of HA, for example sodiumhyaluronate (HA-Na) or potassium hyaluronate (K-HA), and a tetra alkylammonium halide. Tetra alkyl ammonium halides useful in the presentinvention may be represented as below.

where R is C₈H₁₇ to C₁₈H₃₇ and X is Cl or Br.

In the second method, water-insoluble complexes of HA were formed bymodifying HA-Na with organic halides, including but not limited topalmitoyl chloride, acetyl chloride, acetoxyacetyl chloride, ethyl4-chloro-4-oxo-butyrate and acryloyl chloride, in the presence oforganic base HCL acceptors such as triethylamine or pyridine. Thesemodified HA complexes are soluble or partially soluble in certainorganic solvents and mixtures of organic/aqueous solvents.

Method A

The first approach involves an ion-exchange route between the monovalentalkali metal hyaluronate and tetra alkyl ammonium halides, asrepresented above. This reaction occurs by the ion-exchange of thenegatively charged carboxylate groups of the alkali metal hyaluronate,e.g. HA-Na or HA-K, with the positively charged tetra alkyl ammoniumhalides, resulting in the formation of an HA complex with a differencein solubility as compared to unmodified HA. The tetra alkyl ammoniumhalides used in this invention are soluble in both water and organicsolvents, such as alcohols, acetone, 1,4-dioxane and tetrahydrofuran.

Halides that can be used in this invention include, but are not limitedto, tetrabutylammonium bromide, cetyldimethylethylammonium bromide,benzalconium chloride, stearyldimethylbenzylammonium chloride,3-(benzyldimethylammonio)propanesulfonate, benzyldimethyldecylammoniumchloride, benzyldimethyldodecylammonium bromide,benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammoniumchloride, benzyldimethyl(2-hydroxyethyl)ammonium chloride,benzyldimethyltetradecylammonium chloride, and benzethonium chloride.

The preferred tetra alkyl ammonium halide is benzalconium chloride(BzCl), a white or yellowish-white solid in the form of an amorphouspowder or gelatinous pieces. The chemical scheme for forming a complexbetween HA-Na and BzCl is shown below.

BzCl has been investigated previously as an antimicrobial agent andshown to be safe at concentrations up to 0.1% weight. BzCl is a mixturecomprised of alkyldimethylbenzylammonium chlorides.

Three major homologues include C₁₂, C₁₄ and C₁₆ straight chain alkyls.The combination of aliphatic hydrocarbon chains and positively chargedammonium groups imparts facile dissolution in both water and selectorganic solvents.

An added advantage of the present HA complex is that by changing theratio of the hyaluronate, e.g. HA-Na, to BzCl, the solubility of thecomplex can be changed in a buffered solution.

Method B

The second approach involves the covalent modification of the hydroxyland/or carboxylate groups of the monovalent salt, e.g. HA-Na or HA-K,with organic halides in the presence of bases such as triethylamine orpyridine. The chemical scheme for forming a covalent modification ofHA-Na with organic halides is shown below.

The reaction conditions employed results in partial substitution of thehydroxyl or carboxylate groups of HA. This renders the modified HAcomplex insoluble in water and soluble in organic solvents such as DMSOand aqueous/organic mixtures. The preferred organic halides include, butare not limited to, palmitoyl chloride, acetyl chloride, acetoxyacetylchloride, ethyl 4-chloro-4-oxo-butyrate, acryloyl chloride, butyrylchloride, stearoyl chloride, myristoyl chloride, lauroyl chloride,oleoyl chloride, caproyl chloride and decanoyl chloride.

The organic base is selected from the group consisting of triethylamine,pyridine, trimethylamine, lutidine, quinoline, piperidine andpyrrolidine. The choice of the organic halide, organic base, solvent,temperature, and reaction times will determine the degree ofsubstitution and, in turn, the solubility of the modified HA complex inorganic solvents. One skilled in the art, once having the benefit ofthis disclosure, will be able to readily ascertain the particularelements and conditions of such reactions based on the desired result.

The source of HA-Na for either of the two methods listed above can beeither animal-derived or obtained by bacterial fermentation. Thepreferred solvent system for HA complexes prepared according to method Aor B is a blend of organic solvents such as 1,4-dioxane, dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMAC)), N,N-dimethylformamide(DMF), and methylene chloride and water. The HA complex formed accordingto the methods of this invention is not completely soluble in aproticnonpolar solvents or pure water. The preferred solvent system for methodA is a 60/40 blend of 1,4-dioxane/water. The preferred solvent systemfor method B is DMSO using 5 percent by weight tetrabutylammoniumbromide as a phase transfer agent.

In another embodiment of the present invention, the solubility of the HAcomplex can be varied either by changing the ratio of HA-Na to BzCl inmethod A or by changing the acid halide in method B. Complexescontaining 1:1 molar ratios of HA:BzCl ultimately dissolve in aqueousenvironments slower than complexes comprised of 1:0.5 or 1:0.25. At allof these ratios the complexes are less soluble in an aqueous environmentas compared to unmodified HA. The ultimate dissolution of such a complexis primarily through dissociation of HA and BzCl units. By using one ora combination of more than one of the methods described herein, oneskilled in art can appreciate that the dissolution rate of the HAcomplex of the present invention can be tailored to match the kineticsof tissue repair.

The biodegradable, hydrophobic polymer of the present invention isselected from the group consisting of aliphatic polyesters, poly(aminoacids), polyalkylene oxalates, polyamides, tyrosine-derivedpolycarbonates, polyorthoesters, polyoxaesters, poly(anhydrides),polyphosphazenes and biopolymers, e.g. collagen, elastin andbioabsorbable starches and blends thereof. Preferred are the syntheticpolymers.

Currently, aliphatic polyesters are among the preferred synthetic,biodegradable, hydrophobic polymers of the present invention. Aliphaticpolyesters can be homopolymers, copolymers (random, block, segmented,tapered blocks, graft, triblock, etc.) having a linear, branched or starstructure. Suitable monomers for making aliphatic homopolymers andcopolymers may be selected from the group consisting of, but are notlimited to, lactic acid, lactide (including L-, D-, meso and D,Lmixtures), glycolic acid, glycolide, epsilon-caprolactone,para-dioxanone (1,4-dioxan-2-one) and trimethylene carbonate(1,3-dioxan-2-one).

Synthetic, biodegradable, hydrophobic elastomeric copolymers are alsoparticularly useful in the present invention. Suitable elastomericpolymers include those with an inherent viscosity in the range of 1 dL/gto 4 dl/g, preferably about 1 dL/g to 2 dL/g and most preferably about 1dL/g to 1.7 dL/g, as determined at 25° C. in a 0.1 gram per deciliter(g/dL) solution of polymer in hexafluoroisopropanol (HFIP). For thepurpose of this invention, an “elastomeric copolymer” is defined as apolymer that, at room temperature, can be stretched repeatedly to atleast twice its original length and which, upon immediate release ofstress, will return to approximately its original length.

Exemplary synthetic, biodegradable, hydrophobic elastomeric copolymersinclude, but are not limited to, copolymers of epsilon-caprolactone(PCL) and glycolide (including polyglycolic acid (PGA)) with a moleratio of epsilon-caprolactone to glycolide of from about 35/65 to about65/35, more preferably from 45/55 to 35/65; copolymers ofepsilon-caprolactone and lactide (PLA) where the mole ratio ofepsilon-caprolactone to lactide is from about 30/70 to about 95/5 andmore preferably from 30/70 to 45/55 or from about 85/15 to about 95/5;copolymers of para-dioxanone (PDS) and lactide where the mole ratio ofpara-dioxanone to lactide is from about 40/60 to about 60/40; copolymersof epsilon-caprolactone and para-dioxanone where the mole ratio ofepsilon-caprolactone to para-dioxanone is from about 30/70 to about70/30; and blends thereof.

The preferred synthetic biodegradable, hydrophobic polymers andcopolymers for the present invention are 90/10 PGA/PLA, PDS, 65/35PGA/PCL, 60/40 PLA/PCL, 85/15 PLA/PCL, 95/5 PLA/PGA and blends thereof.

The synthetic biodegradable, hydrophobic polymer of the presentinvention can be prepared in the form of a scaffold component by anumber of methods known in the art. If fibrous, the syntheticbiodegradable, hydrophobic polymer may be extruded into fibers and thefibers then may formed into a porous, fibrous nonwoven structure usingstandard wet-lay or dry-lay techniques.

Alternatively, the synthetic scaffold component of this invention can beprepared in the form of a porous, sponge or foam structure by a varietyof techniques well known to those having ordinary skill in the art. Forexample, the hydrophobic polymer may be dissolved in an appropriatesolvent for the polymer and then processed into a foam bylyophilization, supercritical solvent foaming, gas injection extrusion,gas injection molding or casting with an extractable material (e.g.,salts, sugar or similar suitable materials).

In one embodiment, the foam substrate component of the present inventioncan be made by a polymer-solvent phase separation technique, such aslyophilization.

The steps involved in the preparation of these synthetic foams includechoosing the appropriate solvents for the polymers to be lyophilized andpreparing a homogeneous solution of the polymer in the solution. Thepolymer-solvent solution then is subjected to a freezing and a vacuumdrying cycle. The freezing step phase-separates the solution and thevacuum drying step removes the solvent by sublimation and/or drying,thus leaving a porous polymer structure, or an interconnected, open-cellporous foam.

Suitable solvents that may be used in the preparation of the porous foamsubstrate include, but are not limited to, formic acid, ethyl formate,acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers (e.g.,tetrahydrofuran (THF), dimethylene fluoride (DMF), and polydioxanone(PDO)), acetone, methylethyl ketone, dipropyleneglycol methyl ether,1,4-dioxane, 1,3-dioxolane, ethylene carbonate, dimethlycarbonate,benzene, toluene, benzyl alcohol, p-xylene, naphthalene,N-methylpyrrolidone, dimethylformamide, chloroform, 1,2-dichloromethane,dimethylsulfoxide, and mixtures thereof. Among these solvents utilizedin preferred embodiments of the invention, a preferred solvent is1,4-dioxane. A homogeneous solution of the polymer in the solvent isprepared using standard techniques.

In one embodiment, additional solids may be added to the polymer-solventsystem to modify the composition of the resulting foam substrate. As theadded particles settle out of solution, the added solids may be moreconcentrated in desired regions (i.e., near the top, sides or bottom) ofthe resulting tissue implant, thus causing compositional changes in suchregions where desired for a particular purpose.

A variety of types of solids can be added to the polymer-solvent system.Preferably, the solids are of a type that will not react with thepolymer or the solvent. Generally, the added solids have an averagediameter of less than about 1 mm and preferably will have an averagediameter of from about 50 to about 500 microns. When utilized,preferably the solids are present in an amount such that they willconstitute from about 1 to about 50 volume percent of the total volumeof the particle and polymer-solvent mixture (wherein the total volumepercent equals 100 volume percent).

Exemplary solids include, but are not limited to, particles ofdemineralized bone, calcium phosphate particles, calcium carbonateparticles for bone repair, solids that will render the scaffold radioopaque, leachable solids for pore creation, and particles that areeffective as reinforcing materials.

Suitable leachable solids include nontoxic leachable materials such assalts, biocompatible mono and disaccharides, polysaccharides, or watersoluble proteins. The leachable materials can be removed by immersingthe foam substrate with the leachable material in a solvent in which theparticle is soluble for a sufficient amount of time to allow leaching ofsubstantially all of the particles, but which does not dissolve ordetrimentally alter the foam substrate.

Suitable non-bioabsorbable materials include biocompatible metals orbioinert ceramic particles. Further, the non-bioabsorbable materials mayinclude polymers such as polyethylene, polyvinylacetate,polymethylmethacrylate, silicone, polyethylene oxide, polyethyleneglycol, polyurethanes, natural biopolymers, and fluorinated polymers andcopolymers.

In addition, effectors, including drugs, bioactive molecules or cells,such as platelet rich plasma, bone marrow cells, and a combination ofthe two, can be added or injected into the tissue scaffold at the pointof care.

In an alternative embodiment of the invention, the tissue scaffold maybe modified either through physical or chemical means to containbiological or synthetic factors or agents that promote attachment,proliferation, differentiation and matrix synthesis of targeted celltypes. Furthermore, the bioactive factors may also comprise part of thescaffold for controlled release of the factor to elicit a desiredbiological function. Another embodiment would include delivery of smallmolecules that affect the up regulation of endogenous growth factors.Growth factors, extracellular matrix proteins, and biologically relevantpeptide fragments that can be used with the matrices of the currentinvention include, but are not limited to, antibiotics and antiviralagents; anticancer agents; anti-rejection agents; analgesics andanalgesic combinations; anti-inflammatory agents such as acetaminophen;cytostatic agents such as Rapamycin; hormones such as steroids;analgesics; growth factors, including bone morphogenic proteins (BMP-2,BMP-4, BMP-6, BMP-12, BMP-2); sonic hedgehog; growth differentiationfactor (GDF5, GDF6 and GDF8); epidermal growth factor; fibroblast growthfactor; platelet derived growth factor (PDGF); insulin like growthfactor (IGF-I and IGF-II); transforming growth factors (TGF-β I-III);parathyroid hormone, vascular endothelial growth factor (VEGF);genetically engineered cells that express desired proteins; and othernaturally derived or genetically engineered proteins, polysaccharides,glycoproteins, or lipoproteins. The biological factors may be obtainedeither through a commercial source or isolated and purified from atissue.

In yet another alternative embodiment, the tissue scaffolds of thepresent invention can be seeded or cultured with appropriate cell typesprior to implantation. Cells which can be seeded or cultured on thescaffolds of the current invention include, but are not limited to, bonemarrow cells, mesenchymal stem cells, stromal cells, stem cells,embryonic stem cells, chondrocytes, osteoblasts, precursor cells derivedfrom adipose tissue, bone marrow derived progenitor cells, kidney cells,intestinal cells, islets, sertoli cells, peripheral blood progenitorcells, fibroblasts, keratinocytes, nucleus pulposus cells, anulusfibrosis cells, fibrochondrocytes, stem cells isolated from adulttissue, neuronal stem cells, glial cells, macrophages and geneticallytransformed cells or combination of the above cell. The cells can beseeded on the scaffolds of the present invention for a short period oftime, e.g. less than one day, just prior to implantation, or culturedfor a longer (>1 day) period, e.g. greater than one day, to allow forcell proliferation and matrix synthesis within the seeded scaffold priorto implantation.

The scaffold configuration must be effective to facilitate tissueingrowth. A preferred tissue ingrowth-promoting configuration is onewhere the cells of the scaffold are open and of sufficient size topermit cell growth therein. An effective pore size is one in which thepores have an average diameter in the range of from about 20 to about1,000 microns, more preferably, from about 20 to about 500 microns.

The following examples are illustrative of the principles and practiceof the invention, although not intended to limit the scope of theinvention. Numerous additional embodiments within the scope and spiritof the invention will become apparent to those skilled in the art.

EXAMPLE 1

This example describes several procedures used to make HA-BzCl complexesaccording to method A.

Equimolar amounts of sodium hyaluronate (1.5 gm) and benzalconiumchloride (1.3 gm) were mixed together in distilled water. After 1 hourof stirring at 25° C., the precipitated solid was filtered and vacuumdried overnight, yielding 1:1 molar HA-BzCl complex.

In a similar manner, 2:1 and 4:1 molar HA-BzCl complexes were made,except the amount of benzalconium chloride was changed from (1.3 gm) forthe 1:1 molar HA-BzCl complex, to 0.65 gm and 0.33 gm of benzalconiumchloride for the 2:1 and 4:1 molar HA-BzCl complexes, respectively.

An alternate procedure to form 1:1 HA-BzCl complex was also used. Sodiumhyaluronate (1.5 gm) was added to a solution containing benzalconiumchloride (1.3 gm) in a mixture of 1,4-dioxane (118 gm) and water (78.5gm). The solution was stirred for 24 hours at 25° C. and centrifuged for1 hour at a rate of 4,000 rpm using an Eppendorf Centrifuge 5810R.Residual NaCl was removed by dialysis.

EXAMPLE 2

This example describes the process of making a water-insoluble HAcomplex according to method B.

Triethyl amine (8.3 gm) was added to a mixture containing sodiumhyaluronate (500 milligram) in anhydrous N,N-dimethylformamide (250 gm).The mixture was cooled to 0° C. and palmitoyl chloride (23 gm) was addeddrop-wise. The resultant suspension was heated to 45° C. and stirred for20 hours. The suspension was poured into dialysis tubing (12,000-14,000molecular weight cut-off) and dialyzed against methanol and isopropanolfor 2 weeks. Contents inside the dialysis membrane were reduced byrotary evaporation and vacuum dried overnight, yielding 1.5 gm of awhite powder.

EXAMPLE 3

This example describes the stability of the HA-BzCl complex fabricatedaccording to Example 1, in distilled water or aqueous buffer solution.

First, 250 milligrams of 1:1 HA-BzCl complex made in Example 1 wassuspended in 20 grams of distilled water and stirred for 2 weeks at 25°C. The remaining solid was vacuum dried at 40° C. for 48 hours usingphosphorous pentoxide drying agent to yield 55 milligram of a whitesolid. This represents a 78 percent loss of the starting mass of 1:1HA-BzCl complex.

In a second experiment, 72 milligrams of 1:1, 2:1, and 4:1 HA-BzClcomplex made in Example 1 were each suspended in PBS solution (pH 7.4,37° C.) and shaken in a constant temperature water bath. The degradationof the HA-BzCl was monitored by measuring the absorbance peakscorresponding to HA and BzCl at 205 nm and 262 nm, respectively, using aUV/Vis spectrometer. Calibration curves were generated using pure HA andBzCl dissolved in PBS. The results indicated that 100 percent of the 4:1HA-BzCl degraded after 1 day, 100 percent of the 2:1 HA-BzCl complexdegraded after 4 days, and 70 percent of the 1:1 HA-BzCl complexdegraded after 2 weeks.

EXAMPLE 4

This example describes the process of incorporating the HA-BzCl complexdescribed in Example 1 into a porous, foam substrate prepared from ahydrophobic polymer.

The foam substrate was prepared utilizing a copolymer of 65/35 PGA/PCLproduced by Birmingham Polymers Inc. (Birmingham, Ala.) with an I.V. of1.79 dL/g, as measured in HFIP at 30° C. The foam was fabricated using alyophilization procedure as described herein.

A solution of 0.1 weight percent of HA-BzCl solution in a 60/401,4-dioxane/water mixture was prepared.

A laboratory scale lyophilizer (Model Duradry, from FTS Kinetics, StoneRidge, N.Y.), was used in this example. The synthetic foam substrate (70mm×70 mm×2 mm) composed of 65/35 PGA/PCL was placed in a 4-inch×4-inchaluminum mold. The HA-BzCl polymer solution was added to the mold suchthat the solution covered the foam substrate such that the substrate wassubmersed in the solution.

The mold assembly then was placed on the shelf of the lyophilizer andthe freeze-dry sequence begun. The freeze-dry sequence used in thisexample was: 1) −17° C. for 60 minutes; 2) −5° C. for 60 minutes undervacuum 100 mT; 3) 5° C. for 120 minutes under vacuum 20 mT; 4) 20° C.for 120 minutes under vacuum 20 mT.

After the cycle was completed, the mold assembly was removed from thelyophilizer and allowed to degas in a vacuum hood for 2 to 3 hours. Thetissue scaffold comprising the substrate and the incorporated Ha-BZ wastaken from the mold and stored under nitrogen.

FIG. 1 is a scanning electron micrograph (SEM) of a cross-section of thefoam tissue scaffold. The SEM shows the lyophilized HA-BzCl 5incorporated within and dispersed the foam tissue scaffold 10.

EXAMPLE 5

This example describes the process of incorporating the HA-BzCl complexdescribed in Example 1 within a synthetic, porous, nonwoven substrate.

The laboratory scale lyophilizer from Example 4 was used in thisexample. Three sheets of a synthetic nonwoven substrate (70 mm×70 mm×2mm) composed of 90/10 PGA/PCL fibers were prepared and placed in4-inch×4-inch aluminum molds. Three solutions of HA-BzCl in 60/401,4-dioxane/water mixture were prepared with 0.05, 0.10, and 0.25 weightpercent HA-BzCl, respectively. The three solutions were added to thethree molds such that the substrates were immersed in the solutions.

The mold assemblies were placed on the shelf of the lyophilizer and thefreeze-dry sequence begun. The freeze-dry sequence used in this examplewas the same as used in Example 4.

After the cycle was completed, the mold assemblies were removed from thelyophilizer and allowed to degas in a vacuum hood for 2 to 3 hours. Thetissue scaffolds were taken from the mold and stored under nitrogen.

The resulting tissue scaffolds contained the nonwoven substrate havingthe HA-BzCl dispersed there through. FIG. 2 is a scanning electronmicrograph (SEM) of a cross-section of the nonwoven tissue scaffold. TheSEM clearly shows the lyophilized Ha-BzCl 15 incorporated with thesynthetic fibers 20 of the nonwoven substrate.

EXAMPLE 6

This example illustrates that chondrocytes proliferate and synthesize aproteoglycan-rich matrix within the tissue scaffolds described inExample 4. Specifically, the 90/10 PGA/PCL nonwoven scaffolds comprising0.05, 0.1, and 0.25 weight percent HA-BzCl complex in a 60:401,4-dioxane/water mixture from Example 5 were evaluated for chondrocyteresponse.

Primary chondrocytes were isolated from bovine shoulders as described byBuschmann, et al., in J. Orthop. Res., 10, 745, (1992). Bovinechondrocytes were cultured in Dulbecco's modified eagles medium(DMEM-high glucose) supplemented with 10% fetal calf serum (FCS), 10 mMHEPES, 0.1 mM nonessential amino acids, 20 micrograms/ml L-proline, 50micrograms/ml ascorbic acid, 100 U/ml penicillin, 100 microgram/mlstreptomycin and 0.25 micrograms/ml amphotericin B (growth media). Halfof the medium was replenished every other day.

The tissue scaffolds, cut to 5 mm in diameter and 1.5 mm thick, weresterilized for 20 minutes in 70 percent ethanol, followed by five rinsesof phosphate-buffered saline (PBS).

Freshly isolated bovine chondrocytes were seeded at a density of 5×10⁶cells/scaffold in 24 well low cluster dishes, by adding a cellsuspension (15 microliter) onto each scaffold. Cells were allowed toattach to the scaffold for three hours before addition of 1.5 ml ofmedium. Scaffolds were cultured for up to 1 week in a humidifiedincubator at 37° C. in an atmosphere of 5 percent CO₂ and 95 percentair. Half of the medium was replaced every other day.

Scaffolds were harvested at 1 and 7 days, and evaluated for histologicalstaining. At each time point, three samples were fixed in 10 percentbuffered formalin, embedded in paraffin and sectioned using a ZeissMicrotome. Cell distribution within tissuescaffolds was assessed byhematoxylin staining of cross sections of scaffolds 24 hours after cellseeding.

Furthermore, sections were also stained for the presence of sulfatedproteoglycans Safranin-O (SO; sulfated GAG's). Computer images wereacquired using a Nikon Microphot-FXA microscope fitted with a Nikon CCDvideo camera (Nikon, Japan).

FIG. 3 shows SO histological sections (100×) of the 0.05 and 0.1 weightpercent HA-BzCl-impregnated nonwoven tissue scaffolds cultured withbovine chondrocytes for 1 week. The figures show cell attachment,proliferation, and matrix formation in each of the scaffolds.

EXAMPLE 7

This example describes the process of forming a porous, foam tissuescaffold from a solution comprising a blend of HA-BzCl (1:1 molar ratio)complex with a synthetic, biodegradable, hydrophobic polymer.

A solution of 0.10 weight percent of HA-BzCl (1:1) solution in a 60/40dioxane/water mixture was prepared and mixed in a 1:9 volume ratio witha 5% 65/35 PGA/PCL solution in 1,4-dioxane, poured into an aluminum moldand immediately placed over a liquid nitrogen bath for 5 minutes tofreeze the solution. The mold was transferred to a laboratory scalelyophilizer and lyophilized according to the protocol outlined inExample 4.

After the cycle was completed, the mold assembly was removed from thelyophilizer and allowed to degas in a vacuum hood for 2 to 3 hours. Thescaffold was taken from the mold and stored under nitrogen.

The following tables represent the relative solubility behavior of Ha-Bzcomplexes prepared according to the invention in mixtures of1,4-dioxane/water and DMAC/water, respectively. TABLE 1 Room TemperatureSolubility of HA:BzCl in Dioxane/Water. HA:BzCl Mole HA Dioxane/H₂OHa:BzCl Ratio Weight % (w/w) Solubility 1:1 1  0/100 No 1:1 0.5  0/100No 1:1 0.25  0/100 No 1:1 1 50/50 Yes 1:1 1 70/30 No 1:1 1 65/35Partially 1:1 0.5 70/30 No 1:1 0.5 65/35 Partially 1:1 0.5 60/40 Yes 1:10.5 50/50 Yes 1:1 0.25 50/50 Yes 1:1 1 75/25 No 1:1 0.5 75/25 No 1:10.25 75/25 No 1:1 0.25 100/0  No 1:0.5 1  0/100 No 1:0.5 0.5  0/100 No1:0.5 0.25  0/100 No 1:0.5 1 50/50 Yes 1:0.5 0.5 50/50 Yes 1:0.5 0.2550/50 Yes 1:0.5 1 75/25 No 1:0.5 0.5 75/25 No 1:0.5 0.25 75/25 No 1:0.50.25 100/0  NoHA = sodium hyaluronate derived from bacterial fermentation (˜450 000Da)BzCl = benzalconium chloride

TABLE 2 Room Temperature Solubility of HA:BzCl in DMAC/H₂O. HA:BzCl MoleHA DMAC/H₂O HA:BzCl Ratio Weight % (w/w) Solubility 1:1 1 25/75 No 1:10.5 25/75 No 1:1 0.25 25/75 No 1:1 0.25 100/0  No 2:1 1 100/0  No 2:10.5 100/0  No 2:1 0.25 100/0  No 2:1 1 60/40 Yes 2:1 1 50/50 Yes 2:1 0.550/50 Yes 2:1 0.25 50/50 Yes 2:1 1 25/75 No 2:1 0.5 25/75 No 2:1 0.2525/75 No 2:1 0.25  0/100 NoHA = sodium hyaluronate derived from bacterial fermentation (˜450 000Da)BzCl = benzalconium chloride

1. A tissue scaffold suitable for use in repair and/or regeneration ofmusculoskeletal tissue when implanted in a body, comprising: a poroussubstrate comprising a biodegradable, hydrophobic polymer; and ahyaluronic acid complex incorporated with said substrate, wherein saidhyaluronic acid complex is insoluble in water, yet soluble in mixturesof organic and aqueous solvents in which said hydrophobic polymer issoluble.
 2. The tissue scaffold of claim 1 wherein said substrate isselected from the group consisting of a foam and a fibrous, nonwovenmat.
 3. The tissue scaffold of claim 2 wherein said biodegradable,hydrophobic polymer is selected from the group consisting of aliphaticpolyesters, poly(amino acids), polyalkylene oxalates, polyamides,tyrosine-derived polycarbonates, polyorthoesters, polyoxaesters,polyphosphazenes, polyhydroxyalkanoates and biopolymers.
 4. The tissuescaffold of claim 3 wherein said biodegradable, hydrophobic polymercomprises an aliphatic polyester.
 5. The tissue scaffold of claim 4wherein said aliphatic polyester is selected from the group consistingof homopolymers, copolymers and blends of poly(lactic acid),poly(glycolic acid), poly(epsilon-caprolactone), poly(dioxanone) andpoly(trimethylene carbonate).
 6. The tissue scaffold of claim 1 whereinsaid hyaluronic acid complex is a complex of a monovalent alkali metalsalt of hyaluronic acid with a tetra alkyl ammonium halide.
 7. Thetissue scaffold of claim 6 wherein said monovalent alkali metal salt ofhyaluronic acid is selected from the group consisting of sodiumhyaluronate and potassium hyaluronate.
 8. The tissue scaffold of claim 7wherein the molar ratio of said sodium hyaluronate to tetra alkylammonium halide is between about 10:1 and about 1:10
 9. The tissuescaffold of claim 8 wherein said tetra alkyl ammonium halide is selectedfrom the group consisting of tetrabutylammonium bromide,cetyldimethylethylammonium bromide, benzalconium chloride,stearyldimethylbenzylammonium chloride,3-(benzyldimethylammonio)propanesulfonate, benzyldimethyldecylammoniumchloride, benzyldimethyldodecylammonium bromide,benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammoniumchloride, benzyldimethyl(2-hydroxyethyl)ammonium chloride,benzyldimethyltetradecylammonium chloride and benzethonium chloride. 10.The tissue scaffold of claim 5 wherein said hyaluronic acid complexcomprises a complex of sodium hyaluronate with benzalconium chloride ina molar ratio of from about 10:1 to 1:10.
 11. The tissue scaffold ofclaim 10 wherein said hydrophobic polymer is selected from the groupconsisting of a copolymer of glycolide and lactide.
 12. The tissuescaffold of claim 11 wherein said mixture of organic and aqueoussolvents comprises a from about 70 percent to about 45 percent of saidorganic solvent and about 30 to about 55 percent of said aqueoussolvent.
 13. The tissue scaffold of claim 1 wherein said hyaluronic acidcomplex is a complex of sodium hyaluronate with an organic halide. 14.The tissue scaffold of claim 13 wherein said organic halide is selectedfrom the group consisting of palmitoyl chloride, acetyl chloride,acetoxyacetyl chloride, ethyl 4-chloro-4-oxo-butyrate, acryloylchloride, stearoyl chloride, myristoyl chloride, lauroyl chloride,oleoyl chloride, caproyl chloride and decanoyl chloride.
 15. The tissuescaffold of claim 1 further comprising cells seeded or culturedtherewith prior to implantation in said body.
 16. The tissue scaffold ofclaim 15 wherein said cells are selected from the group consisting ofbone marrow cells, mesenchymal stem cells, stromal cells, embryonic stemcells, chondrocytes, osteoblasts, precursor cells derived from adiposetissue, bone marrow-derived progenitor cells, kidney cells, intestinalcells, islets, sertoli cells, peripheral blood progenitor cells,fibroblasts, keratinocytes, nucleus pulposus cells, anulus fibrosiscells, fibrochondrocytes, stem cells isolated from adult tissue,neuronal stem cells, glial cells, macrophages and geneticallytransformed cells.
 17. The tissue scaffold of claim 1 further comprisingbiological or synthetic factors or agents that promote attachment,proliferation, differentiation or matrix synthesis of targeted celltypes.